Cell migration inhibitor

ABSTRACT

An agent for prevention or treatment of vascular disease and renal dysfunction that are accompanied by the migration of vascular smooth muscle cells and mesangial cells comprises as an active ingredient a growth-hormone secretion promoting substance receptor agonist that have a strong inhibitory action on the migration of vascular smooth muscle cells and mesangial cells. Using such an agent for prevention or treatment, one can suppress arteriosclerosis, recurrence of intimal thickening after angioplasty, and even glomerulosclerosis.

TECHNICAL FIELD

This invention relates to a cell migration inhibitor comprising agrowth-hormone secretion promoting substance receptor agonist as anactive ingredient, in particular, an inhibitor for cell migration asinduced by PDGF and angiotensin II, as well as an agent for preventionor treatment of diseases that are accompanied by the migration ofvascular smooth muscle cells or mesangial cells.

BACKGROUND ART

In embryogenesis, cancer, inflammation, etc., cell migration is aphenomenon that precedes the proliferation of the cell. Migration ofvascular smooth muscle cells or mesangial cells also occurs before theirproliferation and is involved with well-known pathologic states,arteriosclerosis and glomerulonephritis.

In normal vascular smooth muscle cells, the greater part of thecytoplasm is occupied by myofilaments and such cells, being responsiblefor vascular constriction and relaxation, are classified as acontractile (differentiated) type. In smooth muscle cells observed infocal arteriosclerosis, the content of myofilaments is decreasedmarkedly whereas organelles such as endoplasmic reticula andmitochondria develop to provide the cells with enhanced ability toproliferate and migrate. Smooth muscle cells of these traits areclassified as a synthetic (proliferative) type. Transformation of smoothmuscle cells to the synthetic type and subsequent abnormal proliferationis hypothesized as a major cause of the progression of arteriosclerosis(Circ. Res. (1986) 58, 427-444). In general, vascular smooth musclecells, when isolated from a living body and cultured, will rapidly losethe traits of the contractile type and change to the synthetic type.

Regarding the onset and development of arteriosclerosis, as well asstenosis and occlusion of blood vessels, the “response-to-injuryhypothesis” proposed by Ross has gained wide acceptance (Nature (1993)362:801-809). According to this theory, a risk factor such ashypertension or hyperlipidemia damages the endothelium and the damagedendothelial cells or inflammatory cells accumulating at the damaged siterelease growth factors such as platelet-derived growth factor (hereunderreferred to as PDGF) and other vasoactive substances includingcytokines, causing the medial smooth muscle cells to migrate to theintima. The migrating smooth muscle cells enhance the proliferativeability and the extracellular matrix-producing activity.

Regimens for preventive drug therapy against arteriosclerosis includeadministering therapeutics for hypertension and hyperlipidemia in orderto reduce the risk factor and administering anti-platelet agents inorder to prevent thrombus formation in arteriosclerotic lesions. Incurrent attempts, drugs that inhibit either one of the abilities ofvascular smooth muscle cells to migrate, proliferate and produce theextracellular matrix are applied to prevent and treat arteriosclerosisand intimal rethickening after angioplasty. For example, tranilast,although weak with its effective concentration being in the range of1×10⁻⁵ M to 1×10⁻³ M, has in vitro capabilities of inhibiting themigration, proliferation and extracellular matrix production of vascularsmooth muscle cells (Atherosclerosis (1994) 107:179-185) and is beingstudied in clinical settings as an agent to prevent restenosis afterpercutaneous transluminal coronary angioplasty (hereunder referred to asPTCA). In addition, C-type sodium peptide, an endogenous vasoactivepeptide has been reported to show a high inhibitory action on themigration of vascular smooth muscle cells in vitro and the peptideitself and the inhibitor of the degradation enzyme hold promise astherapeutics for arteriosclerosis, etc. (Arterioscler. Thromb. Vasc.Biol. (1996) 16:1080-1087).

Mesangial cells originate from arterial smooth muscle cells and theirmigration and proliferation can potentially progress to renaldysfunction. A glomerulus is a network of capillaries in the form of atuft and consists of glomerular endothelial cells, mesangial cells thathold capillaries together, glomerular epithelial cells, glomerular basalmembrane and mesangial matrix. The glomerular construct is broken by anincrease in the mesangial matrix secreted from mesangial cells and thisleads to glomerulosclerosis.

The etiology of glomerulonephritis and the mechanism of its progressionhave not been fully unraveled. One of the theories supported today isthe hypothesis proposed by Floege et al. (Kidney Int. (1993) 39:S47-S54). According to this theory, platelets and macrophages areactivated in glomeruli to release PDGF and basic fibroblast growthfactor, which induce mesangial cell proliferation and resultantglomerulosclerosis. Later studies have confirmed that the proliferationof mesangial cells is preceded by their migration.

The current methods for treating renal dysfunctions such asglomerulonephritis are by no means radical and mostly focus onmaintaining renal functions, reducing urinary protein and controllinghypertension and other complications. Renal dysfunctions have a verydisappointing prognosis and lead to renal failure in a comparativelyshort period; a majority of cases require dialysis therapy andeventually result in death. Hence, the advent of a drug capable ofdrastic treatment is desired.

Growth-hormone secretion promoting substances secrete growth hormonefrom the pituitary gland via growth-hormone secretion promotingsubstance receptors present in the hypothalamus and pituitary. Ghrelinis an endogenous ligand for growth-hormone secretion promoting substancereceptors, discovered from the stomach extract and is found to be inplasma at high concentrations. It has been verified that growth-hormonesecretion promoting substance receptors are also expressed in bloodvessels, heart, lung, pancreas, kidney, liver and fats at the proteinlevel or mRNA level; growth-hormone secretion promoting substance inperipheral organs are being actively studied from physiological andpharmacological aspects (Nature (1999) 402:656-660). It has recentlybeen reported that the expression of growth-hormone secretion promotingsubstance receptors is increased at sites of human coronaryarteriosclerotic lesions but the pathophysiological importance of thephenomenon remains to be determined (Brit. J. Pharmacol. (2001)134:143-149). Rossoni et al. reported that hexarelin, a growth-hormonereleasing peptide, improved the hypercontractile responsiveness toendothelin and hyporesponsiveness to acetylcholine in the blood vascularvessels of hypophysectomized rats (J. Cardiovasc. Pharmacol. (1999)34:454-460). It is suggested that those actions of hexarelin are onvascular endothelial cells. Ghrelin is produced in large amounts in thekidney (FEBS Lett. (2000) 486:213-216) but no report has been made ofthe role (if any) that may be played by ghrelin in the kidney.

DISCLOSURE OF THE INVENTION

An object of the present invention is to discover compounds that have astrong inhibitory action on the migration of vascular smooth musclecells and mesangial cells, thus being able to suppress arteriosclerosis,the intimal rethickening after angioplasty, and even glomerulosclerosis.

The present inventors conducted intensive studies with a view toattaining the stated object. Surprisingly, they found thatgrowth-hormone secretion promoting substance receptor agonists have astrong inhibitory action on the migration of vascular smooth musclecells and mesangial cells. The present invention has been accomplishedon the basis of this finding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the suppressive action of KP-102 on thePDGF-induced migration of human vascular smooth muscle cells;

FIG. 2 is a graph showing the suppressive action of ghrelin on thePDGF-induced migration of human vascular smooth muscle cells;

FIG. 3 is a graph showing the suppressive action of KP-102 and ghrelinon the PDGF-induced migration of rabbit vascular smooth muscle cells;

FIG. 4 is a graph showing the suppressive action of GH, GHRH, hexarelin,MK-0677, NN-703, CP-424,391, S-38855 and GHRP-6 on the PDGF-inducedmigration of human vascular smooth muscle cells;

FIG. 5 is a graph showing the antagonistic action of L-756867 on themigration suppressive action of KP-102, ghrelin and hexarelin on thePDGF-induced migration of human vascular smooth muscle cells;

FIG. 6 is a graph showing the suppressive action of KP-102 on theangiotensin II induced migration of human vascular smooth muscle cells;

FIG. 7 is a graph showing the suppressive action of ghrelin on theangiotensin II induced migration of human vascular smooth muscle cells;

FIG. 8 is a graph showing the suppressive action of KP-102 on thePDGF-induced migration of human mesangial cells; and

FIG. 9 is a graph showing the action of ghrelin on the PDGF-inducedmigration of human mesangial cells.

In the Figures, * and ** denote significant differences at P values of5% and 1%, respectively, to the group treated with a solvent containinga migration stimulant; ## denotes a significant difference between twocompared groups at a P value of 1%.

BEST MODES FOR CARRYING OUT THE INVENTION

According to an embodiment of the present invention, there is provided acell migration inhibitor that comprises a growth-hormone secretionpromoting substance receptor agonist as an active ingredient.

The term “cell migration” as used herein means the migration of cells asinduced by physiologically active substances such as PDGF andangiotensin II that have migration activity. For example, if an isolatedblood vessel is immersed in a PDGF solution, smooth muscle cells comeout to migrate. Cell migration inhibitors inhibit such migration and theinhibitory activity is quantitatively measured by the use of a Boyden'schamber as described in the Example below.

According to another aspect of the invention, there is provided an agentfor prevention or treatment of disease that is accompanied by themigration of vascular smooth muscle cells or a renal dysfunction that isaccompanied by the migration of mesangial cells, characterized in thatit comprises a growth-hormone secretion promoting substance receptoragonist as an active ingredient.

According to still another embodiment of the invention, there isprovided an agent for prevention or treatment of disease that isaccompanied by PDGF- or angiotensin II induced cell migration,characterized in that it comprises a growth-hormone secretion promotingsubstance receptor agonist as an active ingredient.

The term “disease that is accompanied by the migration of vascularsmooth muscle cells” refers to a disease in which for one or otherreason, vascular smooth muscle cells migrate from the media to theintima or intimal vascular smooth muscle cells migrate further towardthe lumen, where they proliferate or produce the extracellular matrix tocause intimal thickening. Examples of such disease includearteriosclerosis obliterans, coronary arteriosclerosis and restenosisafter PTCA. The disease that is accompanied by the migration of vascularsmooth muscle cells is not restricted to blood vessels that governspecified organs. Nor is it restricted to the thickening of arterialvessels and it may include occlusive lesions on venous vessels such asshunted vessels of the veins of patients on dialysis.

The term “angioplasty” as used herein refers to surgery employed in thetreatment of coronary artery disease associated with arterioscleroticlesions including angina pectoris and myocardial infarction and it maybe exemplified by percutaneous transluminal coronary angioplasty,atherectomy, coronary artery bypassing and stent placement.

The term “renal dysfunction that is accompanied by the migration ofmesangial cells” as used herein refers to a condition in which for oneor other reason, mesangial cells migrate to a damaged site, where theyproliferate or produce the mesangial matrix to pressurize glomeruli andimpair their function; examples include renal failure, acuteglomerulonephritis, chronic glomerulonephritis, diabetic nephropathy andnephrosclerosis. The disease under consideration is not restricted toprimary types but may encompass infectious glomerulopathy and othercases of secondary glomerulopathy associated with multi-organ disease.

The term “growth-hormone secretion promoting substance receptor(hereunder GHSR)” as used herein refers to receptors of growth-hormonereleasing peptides and growth-hormone secretagogues and they aretypically responsible for the secretion and release of growth hormone(hereunder GH) in the pituitary gland and as for the GHSRs that areexpressed in other organs (e.g. heart, lung, pancreas, etc.) includingtheir subtypes, their functions are not restricted to known GH secretionor promotion of GH secretion.

The term “growth-hormone secretion promoting substance receptor agonist(GHSR agent)” as used herein refers to a substance that has the activityto act on GHSR and impart some cell response stimulation. Encompassed bythe term are growth-hormone releasing peptides and growth-hormonesecretagogues but it is not restricted to those which are represented byGH secretion or promoted GH secretion. The agent is also not restrictedto substances that act on GHSR invariably expressed in the organs of allspecies of animals and includes substances that act on receptorsexpressed artificially by gene transfer into cell lines.

The term “growth-hormone releasing peptide (hereunder GHRP)” as usedherein means peptides having such a pharmacological activity that theyact on GHSR to promote GH secretion, wherein endogenous peptides as wellas synthetic equivalents and various derivatives thereof are alsoembraced as long as they have comparable activity.

The term “growth-hormone secretagogue (hereunder GHS)” as used hereinrefers to non-peptide substances having such a pharmacological activitythat they act on GHSR to promote GH secretion, wherein variousderivatives thereof are also encompassed as long as they have comparableactivity.

Exemplary growth-hormone secretion promoting substance receptor agonistsinclude GHRPs such as pralmorelin, hexarelin and GHRP-6; synthetic GHSssuch as ibutamorelin methanesulfonate, tabimorelin and capromorelin; invivo GHSR ligands such as ghrelin, GHSR ligand polypeptides (WO01/32705) and adenosine.

Further examples of the growth-hormone secretion promoting substancereceptor agonist are disclosed in the following patents, which areprovided for illustrative purposes only and should not be taken aslimiting.

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If desired, two or more of the growth-hormone secretion promotingsubstance receptor agonists or salts thereof may be employed incombination.

The growth-hormone secretion promoting substance receptor agonists foruse in the invention can be formulated by known pharmaceuticalformulating procedures into various dosage forms such as injection,solution, tablet, powder, subtilized granule, granule and drug deliverysystem (e.g. slow-releasing agent) either alone or in combination withpharmacologically acceptable carriers and additives. The content of thegrowth-hormone secretion promoting substance receptor agonists is notlimited to any particular value as long as the desired effect isobtained.

Examples of the carriers and additives that can be used to formulate theagents for prevention or treatment of the invention include thefollowing which are commonly employed in preparing of medicines: aqueoussolvents such as physiological saline, water (e.g. tap water, distilledwater, purified water and water for injection) and Ringer's solution;non-aqueous solvents such as oily solvents (e.g. vegetable oil) andwater-soluble solvents (e.g. propylene glycol, macrogol, ethanol andglycerin); bases such as cacao buffer, polyethylene glycol,microcrystalline wax, bleached beeswax, liquid paraffin and whitepetrolatum; excipients such as sucrose, starch, mannitol, sorbitol,lactose, glucose, cellulose, talc, calcium phosphate and calciumcarbonate; binders such as cellulose, methyl cellulose, hydroxypropylcellulose, poly(propylpyrrolidone), gelatin, gum arabic, polyethyleneglycol, sucrose and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium hydrogencarbonate, calciumphosphate, carboxymethyl cellulose calcium and calcium citrate;lubricants such as magnesium stearate, talc and sodium lauryl sulfate;flavoring agents such as citric acid, menthol, glycine, sorbitol andorange powder; preservatives and antiseptics such as paraoxybenzoateester, benzyl alcohol, chlorobutanol and quaternary ammonium salts (e.g.benzalkonium chloride and benzethonium chloride); stabilizers such asalbumin, gelatin, sorbitol and mannitol; suspending agents such asmethyl cellulose, poly(vinylpyrrolidone) and aluminum stearate;plasticizers such as glycerin and sorbitol; dispersants such ashydroxypropylmethyl cellulose; solvent promoters such as hydrochloricacid; emulsifiers such as sodium monostearate; and osmolarity modifiersincluding electrolytes such as sodium chloride and non-electrolytes suchas sugar alcohols, saccharides and alcohols.

The pharmaceutical preparations of the invention are usuallyadministered into mammals including human by various means includingintravenous injection, oral application, subcutaneous application, nasalapplication, transpulmonary application and intra-arterial injection.The dosage of the pharmaceutical preparations varies with the patient'sage, body weight, symptoms, the route of administration and otherfactors; for use on adults, pralmorelin, for example, may beadministered in the following manner: ca. 0.1-1000 μg per kg of bodyweight is infused or administered intravenously in one to severalportions per day; alternatively, ca. 10 μg-10 mg per kg of body weightis administered orally in several portions.

The agent for prevention or treatment of the invention can be appliedeither simultaneously with or a certain time before or aftertherapeutics for circulatory disease and renal disease, such as coronarydilators, diuretics, inotropic agents, ACE inhibitors, hypertensiontherapeutics and anti-platelet agents.

The present inventors have found for the first time that growth-hormonesecretion promoting substance or growth-hormone secretion promotingsubstance receptor agonists are effective as drugs that prevent or treatdisease that is accompanied by the migration of vascular smooth musclecells and mesangial cells. The agent for prevention or treatment of theinvention which comprises a growth-hormone secretion promoting substancereceptor agonist as an active ingredient present high migrationinhibitory activity by directly acting on vascular smooth muscle cellsor mesangial cells; it also has low toxicity on various animals andfeature high safety for humans.

The following example and tests are provided for further illustratingthe present invention but are in no way to be taken as limiting.

EXAMPLE 1 Pralmorelin Solution

Pralmorelin dihydrochloride (hereunder KP-102) was dissolved inphysiological saline (Japanese Pharmacopoeia, or JP) and three solutionshaving concentrations of 0.1%, 0.2% and 1.0% were prepared.

[Test 1] Effect of growth-hormone secretion promoting substance receptoragonists on the migration of vascular smooth muscle cells

1. Materials

KP-102 (the compound referred to as GHRP-2 in WO 93/04081 was preparedas a dihydrochloride in the usual manner), ibutamorelin methanesulfonate(as prepared by the method described in WO 94/13696: the compound ofExample 55, hereunder referred to as MK-0677), tabimorelin (as preparedby the method described in WO 97/23508: the compound of Example 1,hereunder referred to as NN-703), capromorelin (as prepared by themethod described in WO 98/58949: the compound of Example 20, hereunderreferred to as CP-424,391), S-38855 (as prepared by the method describedin WO 00/48623: the compound of Example 4), and hexarelin (PENINSULALABORATORIES) were each dissolved in dimethyl sulfoxide (Nacalai Tesque,Inc.) and used after being diluted to specified concentrations withwater for injection (JP, Otsuka Pharmaceutical Co., Ltd.) containing 1mg/mL bovine serum albumin (SIGMA). Human-type ghrelin (PeptideInstitute, Inc.), human-type GHRH (Peptide Institute, Inc.), recombinanthuman growth hormone (Wako Pure Chemical Industries, Ltd.), GHRP-6(BACHEM) and L-756867 (BACHEM) were used after being diluted tospecified concentrations with water for injection containing 1 mg/mLbovine serum albumin. The other reagents used were a smooth muscle cellbasal medium (SmBM of Sanko Junyaku Co., Ltd.), additive factors (SmGM-2additive factor set of Sanko Junyaku Co., Ltd. consisting of a humanepitheliocyte proliferation factor, a human fibroblast growth factor,insulin, fetal bovine serum, gentamicin and amphotericin B), DMEM(GIBCO), fetal bovine serum (GIBCO), trypsin (GIBCO), PDGF (Genzyme) andangiotensin II (SIGMA).

2. Methods

(1) Cultivation of Vascular Smooth Muscle Cells

Human vascular smooth muscle cells were cultured in SmBM with theadditive factors in a culture incubator (37° C., 5% CO₂/95% air) andcells at the 5th-9th passage were used in the test. Rabbit vascularsmooth muscle cells were obtained by an explant technique from the mediaof rabbit thoracic aorta and cultured in DMEM with 10% fetal bovineserum; cells at the 5th-7th passage were used in the test.

(2) Assay of migratory activity

Cells that proliferated to confluence were detached with trypsin, washedtwice with an additive factor free medium, dispersed in SmBM containing0.1% fetal bovine serum and prepared to a cell density of 5×10⁵cells/mL. Migration of cells was assayed according to Koyama et al.method (J. Biol. Chem. (1993) 268:13301-13308) using a 48-well Boyden'schamber (Neuro probe). To be more specific, 28 μL of SmBM containing 10ng/mL of PDGF and 0.1% FBS was placed in the lower chamber and 50 μL ofa cell dispersion preliminarily incubated with each assay compound for10 minutes was placed in the upper chamber. The upper chamber and thelower chamber were separated by a polycarbonate filter (having pores 8μm in diameter) precoated with type I collagen (Nitta Gelatin Inc.)Since the placement of the cell dispersion, the Boyden's chamber wasincubated in a culture incubator (37° C., 5% CO₂/95% air) for 4 hours.The cells on the upper side of the filter were scraped off and the cellsthat had migrated to the lower side of the filter were fixed in methanoland stained with Diff-Quick staining solution (International ReagentsCorporation). The filter had been air-dried on a glass slide and sealedwith Enteran New. The cells were counted under a high-power microscope(×200) in 4 fields per well and the number of cells that had migratedwas averaged to determine the migration activity.

(3) Statistical Analysis

The result of each treated group was represented by the mean±standarddeviation for 3 wells. Statistical significance was estimated by one-wayANOVA, if p value was less than 5%, followed by Dunnett method tocompare between vehicle-treated group and the others in the presence ofa migration inducer. Values of p less than 5% were considered to besignificant.

3. Results and discussion

An insult to the vascular endothelium causes platelets to aggregate andrelease PDGF, etc. PDGF is also secreted by monocyte-derived macrophagesinvading under the injured endothelium. The PDGF stimulates the medialsmooth muscle cells and works as a strong migration factor to promotetheir proliferation, thus playing an important role at the early phaseof arteriosclerosis and the intimal thickening after angioplasty.

The migration of human vascular smooth muscle cells as induced by 10ng/mL of PDGF was significantly suppressed by 1 nM-1000 nM of KP-102,with concentration dependency observed in the range of 1 nM-100 nM (FIG.1). Ghrelin showed a suppressive action between 0.1 nM and 1000 nM,which was concentration dependent in the range of 0.1 nM-100 nM (FIG.2). We also carried out assays using rabbit vascular smooth muscle cellsand found that both KP-102 and ghrelin showed a significant suppressiveaction in the treatment with 10 nM (FIG. 3). Furthermore, we examinedthe efficacy of hexarelin, MK-0677, NN-703, CP-424,391, S-38855 andGHRP-6 which were known growth-hormone secretion promoting substancereceptor agonists on the migration of human vascular smooth musclecells; each compound showed a significant suppressive action at aconcentration of 10 nM. However, no action was exhibited by growthhormone (GH) at 0.1 μg/mL or growth-hormone releasing hormone (GHRH) at10 nM (FIG. 4).

Next, we determined how the GHS receptor antagonist L-756867 affect thesuppressive action of KP-102, ghrelin and hexarelin in the migration ofvascular smooth muscle cells; 1000 nM of L-756867 was found antagonisticto the migration suppressing action of KP-102, ghrelin or hexarelin at10 nM (FIG. 5).

As shown above, the various growth-hormone secretion promoting substancereceptor agonists with different structures suppressed the PDGF-inducedcell migration and lost their action upon treatment with the GHSreceptor antagonist; it is therefore clear that the growth-hormonesecretion promoting substance receptor agonists do not suppress cellmigration as antagonists of the PDGF receptor but suppress cellmigration by a GHSR-mediated mechanism.

The new mechanism of cell migration we found suggested to us thepossibility of suppressing migration induced by other factors. It iswell known that the renin-angiotensin system plays an important role inthe circulation regulatory system. Angiotensin II usually works forvasoconstriction but works for stimulation of migration of vascularsmooth muscle cells if they are transformed to the synthetic type.

The migration of human vascular smooth muscle cells as induced by 100 nMof angiotensin II was significantly suppressed by 0.1 nM-1000 nM ofKP-102 (FIG. 6) and it was also suppressed significantly by 0.1 nM-1000nM of ghrelin (FIG. 7). Thus, it became clear that the growth-hormonesecretion promoting substance receptor agonists can suppress not onlythe migration induced by PDGF but also the migration induced byangiotensin II.

Therefore, the agent for prevention or treatment of the invention whichcomprises the growth-hormone secretion promoting substance receptoragonist as an active ingredient suppresses the migration of vascularsmooth muscle cells and works effectively as an agent for preventing ortreating arteriosclerosis and disease that is accompanied by the intimalthickening after angioplasty.

It should be added that the growth-hormone secretion promoting substancereceptor agonists inhibited not only the migration of human-derivedcells but also the migration of rabbit-derived cells. Hence, themigration inhibitory action of interest is not restricted to humans. Thegrowth-hormone secretion promoting substance receptor agonists are alsoeffective as an agent for preventing or treating arteriosclerosis andother life-style related disease in companion animals.

[Test 2] Effect of Growth-Hormone Secretion Promoting Substance ReceptorAgonists on the Migration of Mesangial Cells

1. Materials and Methods

Human mesangial cells (BioWhittaker) were cultivated in MSGM [basalmedium for mesangial cells (Sanko Junyaku Co., Ltd.) containing an MsGMadditive factor set (Sanko Junyaku Co., Ltd.; consisting of FBS,gentamicin and amphotericin B)] and cells at the 5th-7th passage wereused in the test. The other materials and methods were the same as inTest 1.

2. Results and Discussion

Mesangial cells integrate endothelial cells in a renal glomerulus andregulate the glomerular blood flow by contracting and relaxing. Aninjury or dysfunction of the endothelium activates platelets and theaggregated platelets, as well as the monocyte-derived macrophagesinvading under the endothelium will secrete PDGF. The PDGF works as astrong migration factor for mesangial cells and promotes not only theirproliferation but also the production of the mesangial matrix. Theseactions of PDGF are beneficial to the remodeling of tissues; on theother hand, excessive actions of PDGF may cause glomerulosclerosis.

GHSR has been verified to upregulate in the kidney but its functions areyet to be known. The present inventors therefore studied the action ofgrowth-hormone secretion promoting substance receptor agonists on themigration of mesangial cells. PDGF-induced migration was significantlysuppressed by KP-102 at concentrations of 0.1 nM and more (FIG. 8). Thisaction was also verified in the use of ghrelin (FIG. 9).

Migration is a characteristic phenomenon recognized on mesangial cells.As shown in Test 1, the suppression of migration by growth-hormonesecretion promoting substance receptor agonists is not an antagonisticaction in PDGF receptors but it is a GHSR-mediated event which wouldalso easily suppress the migration due to other migration factors.Therefore, the growth-hormone secretion promoting substance receptoragonists would be effective in the treatment of various renal diseasestriggered by the migration of mesangial cells.

INDUSTRIAL APPLICABILITY

In Test 1, KP-102, hexarelin, GHRP-6, ghrelin, MK-0677, NN-703, CP-424,391 and S-38855 showed potent inhibitory action on the PDGF-inducedmigration of vascular smooth muscle cells and they are in the classes ofGHRP and GHS; at the same time, KP-102, hexarelin, GHRP-6, MK-0677,NN-703 (Eur. J. Endocrinol. (1999) 141:180-189), ghrelin (Nature (1999)402:656-660) and CP-424,391 (Endocrine (2001) 14:121-132) aregrowth-hormone secretion promoting substance receptor agonists. Speakingof L-756867, it is known as a GHS receptor antagonist that binds togrowth-hormone secretion promoting substance receptors (Mol. Endocrinol.(1996) 10:57-61) and suppresses the GHRP- or GHS-stimulated GH secretionfrom pituitary gland cells (J. Endocrinol. (1997) 152:155-158). In Test1, the migration suppressing action of KP-102, ghrelin and hexarelin wasantagonized by L-756867. The growth hormone (GH) and the growth-hormonereleasing hormone (GHRH) showed no action on the PDGF-induced migrationof vascular smooth muscle cells. These facts show that the cellmigration suppressing action of the growth-hormone secretion promotingsubstance receptor agonists is a GHSR-mediated action.

Further, the results of Tests 1 and 2 easily lead us to conjecture thatthe suppressive action of the growth-hormone secretion promotingsubstance receptor agonists on cell migration is not necessarily limitedto vascular smooth muscle cells and mesangial cells but is alsoeffective against the migration of other cells, say, myofibroblastssince vascular smooth muscle cells and mesangial cells becomemyofibroblast-like through transformation. It should be noted thatmyofibroblasts also migrate upon stimulation with PDGF. It has beenpointed out that myofibroblasts are associated with fibrogenesis inorgans as exemplified by cirrhosis, tubulointerstitial disorder andpulmonary fibrosis and thus GHS would also be effective as an agent forpreventing or treating such diseases.

1-15. (canceled)
 16. A method of inhibiting cell migration whichcomprises administering to a patient in need thereof a growth-hormonesecretion promoting substance receptor agonist.
 17. The method of claim16, wherein the cell migration is PDGF-induced cell migration.
 18. Themethod of claim 16, wherein the cell migration is angiotensionII-induced cell migration.
 19. The method in claim 16, wherein the cellmigration is vascular smooth-muscle cell migration.
 20. The method ofclaim 16, wherein the cell migration is mesangial cell migration. 21.The method of claim 16, wherein the cell migration is myofibroblastmigration.
 22. A method of preventing or treating a vascular diseasethat is accompanied by the migration of vascular smooth muscle cellswhich comprises administering to a patient in need thereof agrowth-hormone secretion promoting substance receptor agonist.
 23. Themethod of claim 22, wherein the vascular disease is arteriosclerosis.24. The method of claim 22, wherein the vascular disease is oneaccompanied by the rethickening of the vascular intima afterangioplasty.
 25. A method of preventing of treating renal dysfunctionaccompanied by the migration of mesangial cells which comprisesadministering to a patient in need thereof a growth hormone secretionpromoting substance receptor agonist.
 26. The method of claim 25,wherein the renal dysfunction is glomerulonephritis.
 27. The method ofclaim 26, wherein the glomerulonephritis is mesangial proliferativeglomerulonephritis.
 28. A method of treating or preventing fibrogenesisin organs which comprises administering to a patient in need thereof agrowth-hormone secretion promoting substance receptor agonist.
 29. Themethod of any one of claims 16 to 21, wherein the growth-hormonesecretion promoting substance receptor agonist is a growth-hormonesecretagogue and/or a growth-hormone releasing peptide.
 30. The methodof any one of claims 22-28, wherein the growth-hormone secretionpromoting substance receptor agonist is a growth-hormone secretagogueand/or a growth-hormone releasing peptide.